Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii.

Meiosis is an essential mechanism of gametogenesis for all sexually reproducing species. In vertebrates, one conserved aspect of sex differentiation is that female embryonic germ cells enter meiosis earlier than male germ cells. In some lower vertebrates, female germ cells proliferate prior to entering meiosis, whereas male cells remain in mitotic arrest. Protandrous black porgy fish, Acanthopagrus schlegelii, have a dramatic life cycle involving a characteristic sex change. Black porgy are functional males for their first and second spawning seasons, but approximately half of the fish transform into females during their third year. We cloned the black porgy homologs of dosage suppressor of mck1 homolog (dmc1) and synaptonemal complex protein 3 (sycp3), and examined their expression profiles as well as those of cytochrome P450 family 26 genes (cyp26: cyp26a and cyp26b), retinaldehyde dehydrogenases (raldh: raldh2 and raldh3), retinoic acid receptors (rars: raralpha, rarbeta, rargamma, and rargammab), retinoid X receptors (rxrs: rxralpha, rxrbeta, and rxrgamma) and deleted azoospermia-like (dazl) during gonadal sex differentiation by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Our results show that during gonadal development, germ cells located in ovarian tissue proceed into meiosis earlier than germ cells in testicular tissue. Furthermore, treatment with estradiol-17beta, which induced cyp26 expression, blocked dazl and raldh expression and reduced the expression of rars, rxrs, dmc1, and sycp3. This unique model therefore suggests that the temporal differences in meiosis initiation between females and males are conserved during gonadal sex differentiation in hermaphroditic vertebrates.

Regulation of two forms of gonadotropin-releasing hormone receptor gene expression in the protandrous black porgy fish, Acanthopagrus schlegeli.

Two GnRH receptors (GnRH-R I and GnRH-R II) were obtained in protandrous black porgy (Acanthopagrus schlegeli). We investigated their tissue distribution, developmental/seasonal changes and regulation of expression using in vivo and in vitro (primary cultures of dispersed pituitary cells) approaches. The relative expressions of GnRH-Rs in the pituitary and gonad were as follows: pituitary: GnRH-R I > GnRH-R II; testicular tissue: GnRH-R I > GnRH-R II; ovarian tissue: GnRH-R I = GnRH-R II. GnRH-R I but not GnRH-R II expression was higher in the pituitary during the spawning period as compared to the prespawning. The expression profiles of both forms of GnRH-R were variable in the gonads according to the gonadal stage and season. In vivo, hCG stimulated GnRH-R I and GnRH-R II expression in testis and ovary. The LHRH analog also up-regulated both receptors in testis and but increased only GnRH-R II in the ovary. Sex steroids (estradiol, E2 and testosterone, T) increased the expression of both receptors in the testis and ovary. In the pituitary, sex steroids (E2 and T) increased the expression of GnRH-R I, but not GnRH-II, both in vivo and in vitro. The LHRH analog also specifically up-regulated the expression of GnRH-R I, but not GnRH-R II, by pituitary cells in vitro. All these data suggest that GnRH-R I rather than GnRH-R II may play a major physiological role in the pituitary. In contrast, both GnRH-R I and GnRH-R II may participate in the regulation of gonadal functions, including a possible role during sex change.

Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

DMY gene induces male development in genetically female (XX) medaka fish.

Although the sex-determining gene SRY/Sry has been identified in mammals, homologues and genes that have a similar function have yet to be identified in nonmammalian vertebrates. Recently, DMY (the DM-domain gene on the Y chromosome) was cloned from the sex-determining region on the Y chromosome of the teleost fish medaka (Oryzias latipes). DMY has been shown to be required for the normal development of male individuals. In this study, we show that a 117-kb genomic DNA fragment that carries DMY is able to induce testis differentiation and subsequent male development in XX (genetically female) medaka. In addition, overexpression of DMY cDNA under the control of the CMV promoter also caused XX sex reversal. These results demonstrate that DMY is sufficient for male development in medaka and suggest that the functional difference between the X and Y chromosomes in medaka is a single gene. Our data indicate that DMY is an additional sex-determining gene in vertebrates.

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes.

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.